This project proposes an in depth analysis of innate immune responses of mosquitoes to nicroorganisms, including medically important pathogens and potential bioterrorism agents. A central goal is to understand the immune responses of parasite-susceptible and refractory strains of Anopheles gambiae, the major vector of human malaria, in particular the responses mounted in different organs of the vector during the complex developmental cycle of the parasite in the mosquito. These studies will use the rodent malaria agent, Plasmodium berghei, as a tractable model system. The work will be aided by recent developments which include: (a) full genome sequencing of A. gambiae, and its bioinformatic comparison with the model genome of Drosophila melanogaster, as reported in Science (issue of October 4, 2002); (b) DNA microarrays for RNA profiling in A. gambiae; (c) an efficient reverse genetic method based on direct injection of double stranded RNA in the adult mosquito, permitting functional inactivation of individual genes in vivo; (d) conditional tissue specific transgenic analysis of in vivo gene function; and (e) protocols for immunolocalization that have already permitted the detection of immune stimulated activation of specific genes in specific tissues and cells in the mosquito. A broader analysis of responses to bacteria will facilitate dissection of immune responses, and will place the anti-parasitic responses in context. Mechanisms of molecular recognition leading to immune responses will be of special interest. This work will be followed by (and will facilitate) carefully targeted studies directed towards characterization of mosquito immune responses to the most important human malaria parasite, P. falciparum, which is a considerably more difficult experimental system than P. bergheL Finally, a comparative bioinformatic study of the immune related gene families in two distantly related mosquitoes, A. gambiae and Aedes aegypti will be performed taking advantage of our earlier Anopheles-Drosophila comparison and an ongoing genomic analysis of Aedes, which is an important Iviral vector. This will set the stage for future experimental analysis of immune responses to Aedes- borne pathogens.